Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE A cixiid survey for natural potential vectors of ‘Candidatus Phytoplasma phoenicium’ in Lebanon and preliminary transmission trials R. Tedeschi1,†, L. Picciau1,†,F.Quaglino2,Y.Abou-Jawdah3, M. Molino Lova4,M.Jawhari3, P. Casati2, A. Cominetti2, E. Choueiri5, H. Abdul-Nour6, P.A. Bianco2 &A.Alma1 1 DISAFA, Università degli Studi di Torino, Grugliasco (TO), Italy 2 DISAA, Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Milano, Italy 3 Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon 4 AVSI Lebanon, Centre Jean Paul II, Ghadir, Lebanon 5 Department of Plant Protection, Lebanese Agricultural Research Institute, Zahlé, Lebanon 6 Faculty of Sciences, Lebanese University, Beirut, Lebanon Keywords Abstract 16S rDNA; almond witches’-broom; planthoppers; Prunus sp; weeds. Almond witches’-broom (AlmWB) disease, associated with ‘Candidatus Phy- toplasma phoenicium’, is an emerging threat with real risk of introduction Correspondence in Euro-Mediterranean Countries. Its rapid spread over large geographical R. Tedeschi, DISAFA, Università degli Studi di areas suggests the presence of efficient insect vector(s). In the present work, Torino, Largo P. Braccini 2, 10095 Grugliasco (TO), Italy. Email: [email protected] a survey on cixiids was carried out in Lebanon in the years 2010–2013 in AlmWB-infested almond and nectarine orchards. Insects were collected by †The first two authors contributed equally to the means of different methods, identified with a stereo microscope, and analysed work for phytoplasma identification through 16S rDNA PCR-based amplification and nucleotide sequence analyses. Preliminary transmission trials were performed Received: 25 February 2014; revised version accepted: 28 October 2014; published online: 9 with the most abundant species. A list of the cixiid genera and species present January 2015. in the studied area is given as well as some information about their biology. ‘Ca. Phytoplasma phoenicium’ strains were detected in the genera Cixius, Tachycixius, doi:10.1111/aab.12188 Eumecurus and Hyalesthes. Preliminary trials revealed that Tachycixius specimens were able to transmit the detected strains to healthy peach potted seedlings. Further studies are required to better clarify the taxonomic status and the bio-ethology of collected planthoppers and deeply study their role as phyto- plasma vectors. Introduction The most characteristic symptoms caused by AlmWB on almond trees are (a) shoot proliferation on the main Fruit tree diseases, caused by phytoplasmas, represent trunk with appearance of witches’-broom, (b) devel- an increasing threat in Europe and in the Mediterranean opment of many axillary buds on the branches, with Basin (Janse, 2012). During the last two decades, the out- small and chlorotic leaves, (c) general decline of the break of a lethal devastating almond (Prunus dulcis (Mill.) tree, yield losses and final dieback. A total produce loss D.A.Webb) disease, named almond witches’-broom arises 1–2 years after the initial appearance of the symp- (AlmWB), has led to a rapid decline of almond trees in toms (Abou-Jawdah et al., 2002). Concerning peach and northern Lebanon (Choueiri et al., 2001; Abou-Jawdah nectarine trees, the first symptom observed is the early et al., 2002) and Iran (Salehi et al., 2006). AlmWB was flowering (15–20 days earlier than normal) along with also detected in peach (P. persica) and nectarine (P. persica phyllody, followed by the earlier development of all the var. nucipersica) in southern Lebanon (Abou-Jawdah buds of the infected branches. In addition, several months et al., 2009) and on GF-677 (P. amygdalus × P. persica)in after the typical flowering period, serrate, slim, light Iran (Salehi et al., 2011). green leaves on the plant branches and witches’-brooms 372 Ann Appl Biol 166 (2015) 372–388 © 2015 Association of Applied Biologists Tedeschi et al. Potential cixiid vectors of ‘Ca. Phytoplasma phoenicium’ on the trunk and the crown of the trees are observed Materials and methods (Abou-Jawdah et al., 2009). Diseases similar to AlmWB, Study area inducing axillary proliferation and little yellow leaves in almond trees were reported in Iran (Verdin et al., 2003; The field surveys were conducted during the 4-year Zirak et al., 2009). Interestingly, grafting experiments and period 2010–2013 in two AlmWB-infested orchards molecular analyses revealed that, up to now, AlmWB does of almond and nectarine trees, and surroundings. The not affect plum (P. domestica), apricot (P. armeniaca)and almond 0.2 ha orchard was located in Feghal, district of cherry (P. avium) trees (Abou-Jawdah et al., 2003). Never- Jbeil, in the north of Lebanon at about 165 m a.s.l. The theless, its rapid spread on almond, peach and nectarine 72 almond trees were 10–40 years old. The nectarine orchards confirmed the risk for epidemics in Lebanon 0.4 ha orchard was located in Kfarkela, district of Mar- and in the other Countries of the Mediterranean area. jayoun, in the south of Lebanon at about 600 m a.s.l. Phytoplasmas are wall-less parasitic bacteria living exclu- The 200 nectarine trees were about 10 years old. In sively in the plant phloem as consequence of the transmis- the selected orchards no insecticide treatments were sion by sap-sucking insect vectors (Lee et al., 2000). They performed during the sampling period. are classified in ‘Candidatus Phytoplasma’ species and in taxonomic group/subgroup according to the sequence of Insect collection their 16S ribosomal DNA (16SrDNA) (IRPCM, 2004; Zhao The investigation was carried out by means of yellow et al., 2009). AlmWB is associated with ‘Ca. Phytoplasma sticky traps and Malaise traps. Only one Malaise trap phoenicium’ strains belonging to taxonomic subgroup (165 × 115 × 190 cm) was installed into each orchard 16SrIX-B (Abou-Jawdah et al., 2002; Lee et al., 2012), des- among a group of infected trees in the years 2010–2012. ignated also as 16SrIX-D (Wei et al., 2007; Molino Lova Six double-sided yellow sticky traps (10 × 30 cm) were et al., 2011), and its genetic variants (Molino Lova et al., placed, in each orchard, only during the two-year period 2011). 2011–2012 and were uniformly distributed in the centre The presence and rapid spread of AlmWB in Lebanon of the orchards between infected trees. All sticky traps, entail the activity of one or more vectors. In nature and the Malaise trap jars, were replaced every 2 weeks. phytoplasmas are mainly transmitted by sap-sucking Ethanol 70% was the preservative liquid used for filling insects, mainly Hemiptera Auchenorrhyncha (families the jars. The insect samplings were carried out from the Cicadellidae and Cixiidae) and Sternorrhyncha (Psylli- beginning of February till the end of December in 2010, dae) (Weber & Maixner, 1998; Weintraub & Beanland, while in the following 2 years, in the light of the results 2006). Recent study showed that the leafhopper Asym- obtained in 2010, from the end of March till the end metrasca decedens Paoli plays a major role in spreading of November. Most of the cixiids collected by means of the disease within or to nearby stone fruit orchards Malaise and yellow sticky traps were further analysed for (Abou-Jawdah et al., 2014). Moreover, the presence phytoplasma presence. Additional direct insect samplings of the disease over distantly located regions, and the were performed by means of a sweeping net (35 cm diam- detection of AlmWB phytoplasma in other insect species eter) in spring and late summer 2010 and 2011 and by (Dakhil et al., 2011) may indirectly represent a hypothe- a hand-held mechanical aspirator (D-Vac Vacuum Insect sis that other potential vectors for AlmWB phytoplasma Net-Model 122, Rincon-Vitova Insectaries, Ventura, CA, may be present. Effectively, many phytoplasma dis- USA) in spring 2012 and 2013. These collecting activities eases (i.e. bois noir disease of grapevine) have complex were done in the same orchards previously mentioned epidemiological cycles involving more than one insect and their surroundings on different wild plants present in vector and multiple host plants (Maixner, 2011). As the area. The insects collected in spring 2012 were used some cixiid species (planthoppers) are known to be for controlled transmission trials and then analysed for vector of phytoplasmas infecting many different crops phytoplasma presence. (Alma et al., 2002; Palermo et al., 2004; Weintraub & Beanland, 2006; Jovic´ et al., 2007; Pinzauti et al., 2008), Plant sampling the present work was focused on the survey of the cixiid-fauna present in almond and nectarine orchards of In the spring time of the years 2010–2013, leaf sam- Lebanon with particular attention on their natural infec- ples were collected from 15 almond and 10 nectarine tion by phytoplasmas. Moreover, transmission trials were plants showing typical AlmWB symptoms such as carried out with specimens belonging to the most abun- witches’-broom, phyllody, virescence and chromatic dant genera in order to verify their possible vectoring alterations of the leaves (Abou-Jawdah et al., 2003), activity. and located in the orchard of Feghal and Kfarkela, Ann Appl Biol 166 (2015) 372–388 373 © 2015 Association of Applied Biologists Potential cixiid vectors of ‘Ca. Phytoplasma phoenicium’ Tedeschi et al. respectively. Moreover, leaf and petiole samples were DNA extraction collected from wild plants where Cixiidae specimens had DNA extraction from insects been captured. In particular, samples from 10 and 19 plants of the weed species Smilax aspera L., a monocotyle- Total genomic DNA was extracted from individ- donous plant of the family Smilacaceae, were collected ual plant-hoppers following a protocol adapted in autumn 2011 and in spring 2012, respectively, in the from Marzachì et al. (1998). Briefly, the ethanol- north of Lebanon. In the south, samples from 29 and 11 preserved adults were dried onto filter paper and plants of the weed Anthemis sp., a dicotyledonous plant of homogenised in a CTAB-based buffer (2% w/v cetyl- the family Asteraceae, were collected during spring 2012 trimethyl-ammonium-bromide (CTAB); 1.4 M NaCl; and 2013.